Control of dehydrodiferulate cross-linking in pectins from sugar-beet tissues

Pectins were extracted from roots, petioles and leaves of sugar beet, and cross-linked using hydrogen peroxide and peroxidase. The effects on dehydrodiferulate formation were monitored by HPLC and TLC. Dehydrodimers were formed in different proportions to those found in vivo. There was a net loss of around 50% of the phenolic groups (monomers plus dimers) during dimerisation. Gel filtration showed that root and petiole pectin, but not leaf pectin, increased in molecular weight during cross-linking. The effects of varying the cross-linking conditions were investigated, and it was found that hydrogen peroxide concentration was the most important factor in controlling both the type and amount of dehydrodiferulate formed.     

Control of diferulate formation in dicotyledonous and gramineous cell-suspension cultures

Primary cell wall polysaccharides of some plants carry ester-linked feruloyl groups that can be oxidatively dimerised both within the protoplast and after secretion into the apoplast. Apoplastic dimerisation has been postulated to form inter-polysaccharide cross-links, contributing to wall assembly, but this role remains conjectural. By feeding cultured cells with [¹⁴C]cinnamate, we monitored the kinetics of polysaccharide-binding and subsequent dimerisation of ¹⁴C-labelled feruloyl groups. Cultured maize and spinach cells took up [¹⁴C]cinnamate more rapidly than barley, Arabidopsis, Acer, tomato and rose cultures. Maize and spinach cells rapidly formed [¹⁴C]feruloyl-polysaccharides and, simultaneously, low-M_r [¹⁴C]feruloyl esters. When all free [¹⁴C]cinnamate had been consumed, there followed a gradual recruitment of radiolabel from the low-M_r pool into the polysaccharide fraction. A proportion of the [¹⁴C]feruloyl-polysaccharides was sloughed into the culture medium, the rest remaining wall-bound. Some of the polysaccharide-bound [¹⁴C]feruloyl groups were coupled to form dehydrodiferulates. At least six putative isomers of [¹⁴C]dehydrodiferulate were formed both rapidly (thus intra-protoplasmically) and gradually (thus mainly apoplastically). These data do not support the hypothesis that intra-protoplasmic dimerisation yields predominantly one isomer (8–5′-dehydrodiferulate). In maize, apoplastic coupling was much more extensive in 7-day old than in 2-day-old cultures; indeed, in 2-day-old cultures apoplastic coupling could not be evoked even by exogenous H₂O₂, suggesting strong control of peroxidase action by apoplastic factors. When apoplastic coupling was minimised by exogenous application of peroxidase-blockers (iodide, dithiothreitol and cysteine), a higher proportion of the secreted [¹⁴C]feruloyl-polysaccharides was sloughed into the medium. This observation lends support to the hypothesis that feruloyl coupling contributes to wall assembly.     

Isolation of diferulic bridges ester-linked to arabinan in sugar beet cell walls

After degradation of sugar beet cell walls with Driselase and fractionation of the solubilised products by hydrophobic interaction chromatography, a dehydrodiferuloylated oligoarabinan was isolated. Its structure was assigned to two dimers of (1-->5)-linked arabinose units esterified by a central 8-O-4' ferulic dimer. These results provide the first direct evidence that pectic arabinans in sugar beet cell walls may be covalently cross-linked through dehydrodiferulates.     

Oxidative coupling of a feruloyl-arabinoxylan trisaccharide (FAXX) in the walls of living maize cells requires endogenous hydrogen peroxide and is controlled by a low-M<Subscript>r</Subscript> apoplastic inhibitor

Feruloyl-polysaccharides can be oxidatively coupled in isolated cell walls by peroxidase plus exogenous H₂O₂ in vitro, but the extent to which similar reactions may occur in the apoplast in vivo was unclear. Numerous cellular factors potentially control feruloyl coupling in vivo, and their net controlling influence is not readily studied in vitro. Therefore, we have monitored apoplastic feruloyl coupling in cultured maize cells in vivo using a radiolabelled model substrate, 5-O-feruloyl-α-L-arabinofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-D-xylose (FAXX). FAXX was expected to permeate the wall and to undergo reactions analogous to those normally exhibited by apoplastic feruloyl-polysaccharides in vivo. Little difference was found between the fates of [feruloyl−¹⁴C]FAXX and [pentosyl−³H]FAXX, indicating negligible apoplastic hydrolase or transferase activities. Very little radioactivity entered the protoplasm. Maize cells that had recently been washed in fresh medium were able to bind most of the FAXX (90%) in their cell walls, regardless of the age of the culture. During wall-binding, the [¹⁴C]feruloyl groups were converted to [¹⁴C]dehydrodiferulates and larger coupling products, as revealed by TLC after alkaline hydrolysis. As expected for an oxidative reaction, wall-binding was delayed by added anti-oxidants (ascorbate, ferulate, sinapate, chlorogenate or rutin). It was also completely inhibited by iodide, an H₂O₂-scavenger, indicating a role for peroxidase rather than oxidase. The observations indicate that oxidative coupling of feruloyl groups occurred within the cell wall, dependent on endogenous apoplastic H₂O₂ and wall-localised peroxidase, in vivo. Cells that had not recently been washed in fresh medium were much less able to bind FAXX, indicating the presence in the apoplast of an endogenous inhibitor of oxidative coupling. This inhibitor was of low M_r, was destroyed by heating, and remained in the aqueous phase (pH ≈3.5) when shaken with ethyl acetate. Its effectiveness was not altered by ascorbate oxidase. It is thus a small, heat-labile, hydrophilic inhibitor (not ascorbate) which we suggest plays a natural role in the control of wall cross-linking, and thus potentially in the control of cell growth.